Journal: Nat Commun / Year: 2019 Title: Integrated NMR and cryo-EM atomic-resolution structure determination of a half-megadalton enzyme complex. Authors: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe ...Authors: Diego F Gauto / Leandro F Estrozi / Charles D Schwieters / Gregory Effantin / Pavel Macek / Remy Sounier / Astrid C Sivertsen / Elena Schmidt / Rime Kerfah / Guillaume Mas / Jacques-Philippe Colletier / Peter Güntert / Adrien Favier / Guy Schoehn / Paul Schanda / Jerome Boisbouvier / Abstract: Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely ...Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
BMRB: 34205Combined solid-state NMR, solution-state NMR and EM data for structure determination of the tetrahedral aminopeptidase TET2 from P. horikoshii
Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
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Experimental details
-
Experiment
Experiment
Method
ELECTRON MICROSCOPY
SOLUTION NMR
SOLID-STATE NMR
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
NMR experiment
Conditions-ID
Experiment-ID
Solution-ID
Sample state
Spectrometer-ID
Type
1
1
1
isotropic
1
RFDR 3D
3
2
3
isotropic
1
DARR
2
3
2
isotropic
2
3D 1H-13C NOESY aliphatic
3
4
4
isotropic
2
CCdream (CC)
3
5
4
isotropic
2
NCO
3
6
4
isotropic
2
NCOCX
3
7
4
isotropic
2
NCACX
3
8
4
isotropic
2
CONCACB
3
9
4
isotropic
2
CANCOCX
1
10
5
isotropic
1
hCANH
1
11
5
isotropic
1
hCONH
1
12
5
isotropic
1
hcoCAcoNH
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Sample preparation
Component
Name: tetrahedral amino-peptidase from P. horikoshii / Type: COMPLEX / Details: H78C/H248C mutant / Entity ID: #1 / Source: RECOMBINANT
Molecular weight
Value: 0.49 MDa / Experimental value: NO
Source (natural)
Organism: Pyrococcus horikoshii (archaea)
Source (recombinant)
Organism: Escherichia coli (E. coli)
Buffer solution
pH: 7.4
Specimen
Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Blotting time 2s, force 1, drain time 0.
Details
Type
Solution-ID
Contents
Details
Label
Solvent system
solid
1
20mMTRIS, 50mMsodiumchloride, 100% H2O
protein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 2H15N and 13C at methyls of ILV.
2H15N_ILVCHD2
100% H2O
solid
5
20mMTRIS, 50mMsodiumchloride, 100% H2O
2H13C15N
100% H2O
solid
3
20mMTris, 50mMNaCl, 100% H2O
protein solution at 10 mg/ml was supplemented by methyl-pentanediol, resulting in microcrystalline precipitate. Labeling 13C at all carbons of LKP residues
LKP-labeled
100% H2O
solid
4
20mMTRIS, 50mMsodiumchloride, 100% H2O
H2O
13C15NTET2
100% H2O
solution
2
50mMTris, 50mMNaCl, 100% D2O
2H15N_ILV-CH3
100% D2O
Sample
Conc. (mg/ml)
Component
Isotopic labeling
Solution-ID
20mM
TRIS
none
1
50mM
sodiumchloride
none
1
20mM
TRIS
none
5
50mM
sodiumchloride
none
5
20mM
Tris
none
3
50mM
NaCl
none
3
20mM
TRIS
none
4
50mM
sodiumchloride
none
4
50mM
Tris
none
2
50mM
NaCl
none
2
Sample conditions
Conditions-ID
Ionic strength
Label
pH
Pressure (kPa)
Temperature (K)
Temperature err
1
50mM
MAS_50kHz
7.5
1atm
300K
2
3
50mM
15kHzMAS
7.5
1atm
300K
2
50mM
solution
8
1atm
328K
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Data collection
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
Microscopy
Model: FEI POLARA 300
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 20000 X / Calibrated magnification: 25773 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 3500 nm / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holder
Cryogen: NITROGEN Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recording
Average exposure time: 4 sec. / Electron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 90
Image scans
Movie frames/image: 40
NMR spectrometer
Type
Manufacturer
Model
Field strength (MHz)
Spectrometer-ID
Details
Bruker AVANCE II
Bruker
AVANCEII
600
1
solid-state NMR
Varian INOVA
Varian
INOVA
800
2
solution-NMR NOEs
-
Processing
EM software
ID
Name
Version
Category
1
RELION
1.4
particleselection
2
Latitude
3
imageacquisition
4
CTFFIND
3
CTFcorrection
7
iMODFIT
1.44
modelfitting
9
RELION
1.4
initialEulerassignment
10
RELION
1.4
finalEulerassignment
11
RELION
1.4
classification
12
RELION
1.4
3Dreconstruction
13
X-PLOR
4.39
modelrefinement
CTF correction
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
Num. of particles selected: 30407
Symmetry
Point symmetry: T (tetrahedral)
3D reconstruction
Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27130 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model building
Protocol: OTHER
NMR software
Name
Developer
Classification
CcpNmr Analysis
CCPN
refinement
CYANA
Guntert, MumenthalerandWuthrich
structurecalculation
CcpNmr Analysis
CCPN
chemicalshiftassignment
CcpNmr Analysis
CCPN
peakpicking
Xplor-NIH
Schwieters, Kuszewski, TjandraandClore
structurecalculation
NMR ensemble
Conformers submitted total number: 10
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